Project Summary Neutrophils protect against infection but also mediate inflammatory tissue injury. As a result, targeting neutrophils therapeutically will require a detailed understanding of their basic biology, focused on domains wherein the defensive and pathogenic functions of neutrophils may diverge. In the first cycle of the present award, we explored two related GPI-linked neutrophil surface proteins of poorly-characterized function, Ly6G in mice and CD177 in humans. We showed that both proteins associate at a molecular level in cis with neutrophil surface ?2 integrins and that their ligation thereby attenuates neutrophil migration. Taking advantage of the experimental potential of murine inflammatory models, we found that Ly6G ligation selectively reduced integrin-mediated migration typical for neutrophil diapedesis toward sterile triggers, leaving integrin-independent migration to infectious stimuli largely unperturbed. Our preliminary data now show evidence that Ly6G differentiates subphenotypes within murine neutrophils, while in humans CD177pos and CD177neg neutrophils differ in gene expression and potentially in cytokine production. Together with the productivity of the first cycle of the award, these findings support deeper investigation of the role of these Ly6-family molecules in neutrophil biology. We propose two new and independent specific aims. Aim I pursues the mechanisms by which Ly6G and CD177 alter neutrophil ?2 integrin function, including a search for novel endogenous counterligands. Aim II develops our preliminary RNAseq data distinguishing neutrophil subtypes based on expression of Ly6G and CD177, both from healthy donors and from adults and children with inflammatory arthritis and the transient but intensely inflammatory vasculitis Kawasaki disease. Together, these studies will advance the understanding of neutrophils by defining how Ly6 family members regulate ?2 integrins to control cell migration and identifying neutrophil phenotypes reflected in differential expression of Ly6G and CD177.